BBA - Bioenergetics 1860 (2019) 148053

BBA - Bioenergetics 1860 (2019) 148053 Contents lists available at ScienceDirect BBA - Bioenergetics journal homepage: www.elsevier.com/locate/bbabio Pigment-protein complexes are organized into stable microdomains in T cyanobacterial thylakoids ⁎ A. Strašková1, G. Steinbach1,2, G. Konert, E. Kotabová, J. Komenda, M. Tichý, R. Kaňa Institute of Microbiology, Czech Academy of Sciences, Centre Algatech, Novohradská 237, 379 81 Třeboň, Czech Republic ARTICLE INFO ABSTRACT Keywords: Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are Photosynthesis conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the inter- Confocal microscopy actions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that Photosystems have been defined as a domain-like structures characteristic by different biochemical composition andfunction Thylakoid membrane (Albertsson P-Å. 2001,Trends Plant Science 6: 349–354). We explored this complex organization of thylakoid Membrane heterogeneity Microdomains pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images Cyanobacteria captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem Population heterogeneity I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These micro- compartments are of sub-micrometer in sizes (~0.5–1.5 μm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evo- lutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids. 1. Introduction Chloroplast represents other bioenergetics organelle of higher plants and algae. Light-photosynthetic reactions proceed on thylakoid mem- Biological membranes were originally described as a fluid mosaic brane that is heterogeneously structured into stacked and unstacked with uniform distribution of proteins and lipids [1]. Later, hetero- regions defined as granal and stromal thylakoids (see e.g. themost geneous membrane areas were found in a form of lipid rafts in animal recent reviews [16–18]). Grana are stabilized by physicochemical cells [2], various bacterial microdomains (MDs) [3–5] or raft-like MDs forces [19,20] controlled by ion compartmentation [21]. All primary in mitochondria [6]; it led to a change in the paradigm of membrane photochemical reactions, including light-harvesting, charge separation organization proposing mosaic macrostructure of cellular membranes and subsequent electron transport processes are catalyzed by hetero- with specific MDs [7]. Recently, a heterogeneous distribution of geneously distributed membrane proteins complexes of Photosystem I membrane proteins has been intensively discussed also in plant cyto- (PSI), Photosystem II (PSII) and cytochrome b6f complex. There is a plasmic membranes [8,9] or in plant mitochondrial membrane [10]. clear lateral asymmetry between granal/stromal thylakoids that form The mitochondrial enzymes of oxidative phosphorylation (OXPHOS) (micro)domains with different biochemical composition and function are known to form heterogeneous membrane compartments [11–13] [22]. Typically, a higher PSII/PSI ratio is typical for granal (stacked) that highly restrict the diffusion of OXPHOS enzymes [14]. Therefore, a and lower for stromal (unstacked) thylakoids [22]. In higher plants, the “plasticity model” of inner mitochondria membranes has been sug- light-harvesting efficiency of photosystems is increased by their mem- gested [15]. brane embedded pigment-protein antennas of photosystems (e.g. ⁎ Corresponding author. E-mail address: [email protected] (R. Kaňa). 1 A. Strašková and G. Steinbach contributed equally to this study. 2 Present address: Institute of Biophysics, Biological Research Center, Szeged, Hungary. https://doi.org/10.1016/j.bbabio.2019.07.008 Received 5 December 2018; Received in revised form 28 June 2019; Accepted 18 July 2019 Available online 22 July 2019 0005-2728/ © 2019 Published by Elsevier B.V. A. Strašková, et al. BBA - Bioenergetics 1860 (2019) 148053 LHCII). PsaF-YFP fusion at the C-terminus of PsaF, pUC18:psaF-YFP-CmR It contrasts with cyanobacteria in which the light-harvesting an- plasmid was used to transform the Synechocystis WT strain as described tenna complexes, Phycobilisomes (PBS)[23] are situated on the thy- in [40]. This plasmid was constructed by insertion of the YFP coding lakoid membrane surface and can form huge functional megacomplexes sequence (corresponding to a Venus sequence, [43]) at the end of the 3′ with both photosystems [24]. In fact, cyanobacteria represent an evo- coding region of the psaF gene (sll0819). As a selectable marker lutionary ancestor of plant chloroplasts [25,26]. Their thylakoids chloramphenicol-resistance gene (CmR) was inserted downstream of the harbor components of respiratory electron transport chain [27] and psaJ (sml0008) gene which is located downstream the psaF gene in the their thylakoids show no signs of the membrane stacking [28,29]. This Synechocystis genome. Transformants were selected on BG11 plates makes cyanobacteria a unique model system to study localization of with 5 μg/mL chloramphenicol, genome copies were segregated by thylakoid protein complexes independently of membrane stacking. In plating on the chloramphenicol concentration up to 40 μg/mL. PCR was vivo microscopic data have suggested a heterogeneous distribution of used to show integration of YFP and elimination of the WT gene copies. pigment-protein complexes (PPCsincluding PSI, PSII and PBS) in thy- The strains were cultivated on a rotary shaker under moderate light lakoids of various cyanobacterial strains including Synechocystis sp., conditions (white light, 40 μmol of photons m−2 s−1, 30 °C) in liquid Synechococcus sp. or Anabaena sp. Based on these data, several distinct BG11 medium. models of PPCs organization in cyanobacterial thylakoids were built Accession Numbers: Sequence data from this article can be found in proposing either radial (i.e. with variability between inner/outer thy- the GenBank/EMBL databases under the following accession numbers: lakoid layers [30–32]) or lateral [33,34] heterogeneity in PPCs, espe- PsaF (Sll0819), BAA18108; and PsaJ (Sml0008). cially photosystems, composition. Different methods used in these studies also led to different conclusions in respect to the photosystems 2.2. Analysis of protein complexes location: PSI has been preferentially localized either to the outermost (see electron microscopic data in [32]) or to the inner membrane thy- Thylakoid membranes were prepared by breaking cells with zir- lakoids (see hyperspectral confocal fluorescence image data in[31]). konia/silica beads using Mini-Beadbeater (BioSpec Products, USA) as Moreover, in vitro AFM experiments with isolated thylakoid membrane described by [44]. The protein composition of cyanobacterial mem- proposed existence of a specific type of small PSI MDs in Synechocystis branes was analyzed by two-dimensional polyacrylamide gel electro- sp.PCC 6803 [35]; electron microscopy pictures showed arrays of PSII phoresis (PAGE) combining clear native electrophoresis (CN-PAGE) in the same organism [36]; a specific bioenergetics MDs were re- with denaturing SDS-PAGE. CN-PAGE was performed in 4–14% gra- cognized by confocal microscopy in Gloeobacter violaceus [37], a pri- dient polyacrylamide gel (acrylamide to BIS-acrylamide ratio was 1:60) mitive thylakoid-less cyanobacterium [38]. These results clearly according to [45] with modifications described in [46]. Native gels showed non-existence of a conclusive model for the thylakoid mem- were photographed and scanned for chlorophyll fluorescence. In- brane organization of PPCs in cyanobacteria. dividual proteins in membrane complexes separated by CN-PAGE were In the present work we addressed complex organization of thylakoid resolved in the second dimension by SDS-PAGE in a denaturing 12–20% membrane proteins by a simultaneous in vivo detection of all major linear gradient polyacrylamide gel containing 7 M urea. Separated PPCs (PSI, PSII, PBS) by means of 3D confocal imaging. Our data proved proteins were further visualized by staining with Coomassie Blue heterogeneous organization of these PPCs into microdomains (MDs) (Quick Coomassie Stain; Generon, Ltd., GB) and the identity of the that define mosaic like structure of thylakoid membrane of stained PSI proteins was verified by mass spectrometry as described in Synechocystis sp. PCC 6803 strain. This conclusion is based on following [47]. methods and results: (1) Simultaneous three channel localization of PPCs [39] has been adapted for 3D imaging of the whole cyanobacterial 2.3. Measurements of PSI activity thylakoid; (2) YFP tagging of PSI [40] allowed localization

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